A Rho family small GTP-binding protein (hereinafter, may be simply referred to as a Rho family protein) belongs to one group of small GTP-binding proteins (hereinafter, may be simply referred to as a small G protein). A small G protein works as a signal amplifier between a cell membrane receptor, and an effector participating in an intracellular signal transduction pathway. Further, the small G protein specifically binds to guanosine 5′-triphosphate (GTP) or guanosine 5′-diphosphate (GDP), and shows an enzyme activity of hydrolyzing the bound GTP to GDP. When an extracellular signaling substance binds to a cell membrane receptor, its signal is transduced to a small G protein, which subsequently leads to a reaction exchanging a GDP bound to the small G protein for an intracellular GTP (hereinafter, the reaction may be abbreviated to GDP/GTP exchange reaction). Consequently, an active (GTP binding form) small G protein is generated. The active small G protein acts on the effector to amplify the signal. Then, the active small G protein hydrolyzes the bound GTP to GDP by its enzyme activity, and thereby becomes inactivated. Thus, the active small G protein works as a molecular switch in an intracellular signal transduction pathway by exchanging the guanine nucleotides.
Cdc42, Rac1, RhoA and the like are known as Rho family proteins. Cdc42 regulates filopodia formation in a fibroblast. Rac1 regulates superoxide production in leukocytes and macrophages, while it regulates cell membrane ruffling and lamellipodia formation in fibroblasts. Further, Cdc42 and Rac1 are capable of activating the c-Jun N-terminal kinase signal transduction pathway. Thus, Rho family proteins are involved in various kinds of cell function by regulating the intracellular signal transduction. For example, cytoskeleton restructuring, cell adhesion, gene expression, and the like are known as a Rho family protein-mediated cell functions. Such functions mediated by Rho family proteins are considered to regulate morphogenesis during ontogeny, migration of leukocytes and the like, axon degeneration, tumor metastasis, and tumor invasion.
Rho guanine nucleotide exchange factor (hereinafter, may be abbreviated as Rho-GEF) is a member of a family of proteins involved in the molecular switching of a Rho family protein. Rho-GEF can function to accelerate the GDP/GTP exchange reaction of a Rho family protein, and can thereby accelerate the activation of the Rho family protein. Rho-GEF plays an important role through this function, in regulating Rho family protein-mediated intracellular signal transduction. Hereinafter, the function of accelerating the GDP/GTP exchange reaction may be referred to as GEF activity.
Rho-GEF has a characteristic domain structure, such as a Db1 homology domain (hereinafter, may be abbreviated as DH domain) and a pleckstrin homology domain (hereinafter, may be abbreviated as PH domain). The DH/PH tandem structure is a typical domain structure for Rho-GEF. Hereinafter, the tandem structure of DH domain and PH domain may be referred to as DH/PH domain.
The DH/PH domain is an important domain participating in the Rho-GEF-mediated activation of a Rho family protein, and is considered to be an active domain of Rho-GEF. For example, it has been reported that a protein, which comprises a C-terminal region of the amino acid sequence of proto-Db1, and contains a DH/PH domain, activated a Rho family protein (Non-patent Reference 1). proto-Db1 is a prototype of Rho-GEF. Specifically, this report showed that a protein consisting of a C-terminal region within the entire amino acid sequence of Proto-Db1 with 925 amino acids, which was generated by deletion of the N-terminal amino acid residues from the 1st to the 497th, activated a Rho family protein, and consequently participated in cellular morphological change. From these facts, the activation of proto-Db1 is considered to be an oncogenic activation. Hereinafter, a protein that consists of the C-terminal region of proto-Db1 is referred to as an oncogenic-Db1. It has been reported that oncogenic-Db1 bind to RhoA, Cdc42 and Rac1, and that they have a GEF activity for Cdc42 and RhoA while they do not exert a GEF activity to Rac1 (Non-patent Reference 2).
Vav (Non-patent Reference 3 and 4), ost (Non-patent Reference 5), 1bc (Non-patent Reference 6), and the like, are known to be genes that encode proto-Db1 family proteins. These genes are known to be involved in cancer. Further, trio (Non-patent Reference 7), kalirin (Non-patent Reference 8), and the like, are reported to be genes that encode proteins working as Rho-GEF. A trio knocked-out mouse shows an abnormal skeletal muscle structure and an abnormal brain structure in embryogenesis. Kalirin is involved in axon formation in neurons. Thus, each protein working as a Rho-GEF is involved in a cellular function particular to each kind of protein, and activates a different Rho family protein.
Literatures referred in the present description are listed hereunder.
Non-patent Reference 1: Bi, F. et al., Molecular and Cellular Biology, 2001, Vol. 21, p. 1463-1474
Non-patent Reference 2: Hart, M. J. et al., Journal of Biological Chemistry, 1994, Vol. 269, p. 62-65
Non-patent Reference 3: Katzav, S. et al., EMBO Journal, 1989, Vol. 8, p. 2283-2290
Non-patent Reference 4: Costello, P. S. et al., Proceedings of The National Academy of Sciences of The United States of America, 1999, Vol. 96, p. 3035-3040
Non-patent Reference 5: Horii, Y. et al., EMBO Journal, 1994, Vol. 13, p. 4776-4786
Non-patent Reference 6: Toksoz, D. et al., Oncogene, 1994, Vol. 9, p. 621-628
Non-patent Reference 7: O'Brien, S. P. et al., Proceedings of The National Academy of Sciences of The United States of America, 2000, Vol. 97, p. 12074-12078
Non-patent Reference 8: Penzes, P. et al., Journal of Neuroscience, 2001, Vol. 21, p. 8426-8434
Non-patent Reference 9: Sambrook et al., Eds., “Molecular Cloning, A Laboratory Manual, 2nd Edition”, 1989, Cold Spring Harbor Laboratory
Non-patent Reference 10: Muramatsu Masari., Ed., “Labomanual Genetic Engineering”, 1988, Maruzen Co., Ltd.
Non-patent Reference 11: Mading, K. et al., Proceedings of The National Academy of Sciences of The United States of America, 2000, Vol. 97, p. 559-564
Non-patent Reference 12: Ulmer, K. M. Science, 1983, Vol. 219, p. 666-671
Non-patent Reference 13: Ehrlich, H. A., Ed., PCR Technology. Principles and Applications for DNA Amplification, 1989, Stockton Press
Non-patent Reference 14: Saiki, R. K. et al., Science, 1985, Vol. 230, p. 1350-1354
Non-patent Reference 15: Jikken Igaku (Experimental Medicine), 1994, Vol. 12, No. 6, p. 35
Non-patent Reference 16: Frohman, M. A. et al., Proceedings of The National Academy of Sciences of The United States of America, 1988, Vol. 85, No. 23, p. 8998-9002
Non-patent Reference 17: Sanger, F. et al., Proceedings of The National Academy of Sciences of The United States of America, 1977, Vol. 74, p. 5463-5467
Non-patent Reference 18: Maxam, A. M. et al., Methods in Enzymology, 1980, Vol. 65, p. 499-560
Non-patent Reference 19: Ohara, O. et al., DNA Research, 1997, Vol. 4, p. 53-59